Bcl-2 is known to prevent apoptosis in response to a variety of stimuli. It is said to work through binding to proapoptotic Bcl-2 family members such as Bax and Bad. However, its precise mechanism of action is poorly defined. We have analyzed Bcl-2 function in the prevention of apoptosis from chemotherapeutic agents and other stimuli. We have found that Bcl-2 function can be altered by phosphorylation. Agents that interfere with microtubule function initiate a signalling cascade that among other things, activates jun N-terminal kinase or JNK, which appears to be involved in Bcl-2 phosphorylation. The site on Bcl-2 that is phosphorylated with attendant inhibition of Bcl-2 function is serine 70. A Bcl-2 mutant that lacks serine 70 or its adjacent loop region is hyperfunctional and cannot be inhibited by phosphorylation. We have also found that apoptosis from paclitaxel and vincristine is mediated substantially through the fas/fas ligand pathway. Bcl-2 interferes with paclitaxel-induced apoptosis by blocking the upregulation of fas ligand expression in the tumor cells. It does this by binding to calcineurin and preventing its activation. Thus, the transcription factor, NFAT, remains phosphorylated and nonfunctional and is unable to translocate to the nucleus to induce fas ligand expression. Bcl-2 binds to calcineurin through its BH4 domain. Another unexpected finding in this work was that Bcl-2 appears to be capable of preventing the generation of nitric oxide, an important early step in some forms of apoptosis. Future studies are aimed at understanding the mechanism of Bcl-2 inhibition of nitric oxide generation. - fas/fas ligan, NFAT, calcinerurin, nitric oxide